Download NZYTaq with 5× Gel Load Reaction Buffer

Magnesium Chloride solution: the provided 50 mM
MgCl2 solution allows users to optimize MgCl2
concentrations. Vortex the MgCl2 solution thoroughly after
thawing and prior to use.
NZYTaq with 5× Gel Load
Reaction Buffer
Catalogue number:
MB03801, 500 U
MB03802, 1000 U
MB03803, 2500 U
Description
NZYTaq DNA polymerase is a recombinant modified form of
Taq DNA polymerase purified from Escherichia coli. NZYTaq
is provided with 5× Gel Load Reaction Buffer allowing
reactions to be loaded directly into gels without the extra
adding of loading dye. This Gel Load Reaction Buffer is
composed by a blue and yellow dye. The blue dye migrates
at the same rate as a 3-5 kb DNA fragment in a 1% (w/v)
agarose gel. The yellow dye migrates at a rate faster than
primers (<50 bp) in a 1% (v/v) agarose gel. The 5× Gel Load
Reaction Buffer is not suitable when direct fluorescent or
absorbance readings are required without prior purification
of the amplified DNA from PCR. NZYTaq DNA polymerase
lacks 3´5´ exonuclease activity. Resulting PCR products
have an A overhang and are suitable for cloning with
NZYTech´s TA PCR cloning kits (MB053 or MB137).
Storage temperature
NZYTaq DNA polymerase should be stored at -20 °C in a
constant temperature freezer. NZYTaq DNA polymerase will
remain stable up to 3 years if stored as specified.
Storage buffer
20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM
DTT, 50% (v/v) glycerol.
Standard Protocol
The following standard protocol serves as a general guideline
and a starting point for any PCR amplification. Optimal
reaction conditions (incubation times and temperatures,
concentration of DNA Polymerase, primers, MgCl2, and
template DNA) vary and may need to be optimized.
1. On ice, in a sterile, nuclease-free microcentrifuge tube,
prepare a reaction mixture for the appropriate number of
samples to be amplified. A single reaction mixture should
combine the following components (for a 50 μL reaction):
5× Gel Load Reaction Buffer
(provided)
10 μL
MgCl2, 50 mM (provided)
1.5-4.0 mM
dNTPs mix
0.25-0.5 mM
Primers
0.2-0.5 μM
Template DNA
0.5 μg - 0.1 ng
NZYTaq DNA polymerase (5 U/μL)
0.25-1 μL
Nuclease-free water
up to 50 μL
2. If using a thermal cycler without a heated lid, overlay the
reaction mix with 1-2 drops of mineral oil to prevent
evaporation during thermal cycling. Centrifuge the reactions
in a microcentrifuge for 5 seconds.
3. Perform PCR using standard parameters.
Cycle step
Temp.
Time
Cycles
Initial
denaturation
95 ºC
120 s
1
Denaturation
95 ºC
30-60 s
Unit definition
Annealing
*
30-60 s
One unit is defined as the amount of enzyme required to
catalyse the incorporation of 10 nmoles of dNTPs into acid
insoluble material in 30 minutes at 72 °C, under the following
assay conditions: 50 mM Tris-HCl, pH 9.0, 50 mM NaCl, 2.5
mM MgCl2, 200 μM each of dATP, dCTP, dGTP, dTTP (a mix
of unlabelled and α-32P-labelled), 10 μg activated salmon
sperm DNA in a final volume of 50 μL.
Extension
72 ºC
30-60 s/kb
Final Extension
72 ºC
5-10 min
Enzyme concentration: 5 U/μL
Gel Load Reaction Buffer (5×): Proprietary formulation
supplied at pH 8.8. Vortex the reaction buffer thoroughly
after thawing and prior to use. Repeated freeze-thaw cycles
will affect the stability of the buffer (the buffer will remain
stable at 4 °C for at least one month).
25-35
1
*Annealing temperature should be optimized for each primer set
based on the primer Tm; typically it should be Tm-5 ºC.
4. Separate PCR products by agarose gel electrophoresis
(0.7-1.2%, w/v) and visualize with Greensafe Premium
(MB132) or any other means.
Primer Design
PCR primers generally range in length from 15–30 bases and
are designed to flank the region of interest. Primers should
contain 40–60% GC, and care should be taken to avoid
sequences that might produce internal secondary structure.
The 3´-ends of the primers should not be complementary to
avoid the production of primer-dimers. Primer-dimers
unnecessarily delete primers from the reaction and result in
an unwanted polymerase reaction that competes with the
desired reaction. Avoid three G or C nucleotides in a row near
the 3´-end of the primer, as this may result in non-specific
primer annealing, increasing the synthesis of undesirable
reaction products. Ideally, both primers should have nearly
identical melting temperatures (Tm); in this manner, the two
primers should anneal at roughly the same temperature.
DNA template
Functional assay
5× Gel Load Reaction Buffer is tested for performance in a
polymerase chain reaction (PCR) using NZYTaq DNA
Polymerase for the amplification of a 1000 bp fragment of
the glucokinase gene from Escherichia coli genomic DNA.
The resulting PCR product is visualized as a single band in a
GreenSafe stained agarose gel.
Troubleshooting
No product amplification or low yield
The optimal amount of starting material may vary depending
on the template DNA quality and complexity. In general, we
recommend using 500-50 ng of genomic DNA template.
Lower amounts of DNA template (typically 50-1 ng) can be
used for amplification of lambda or plasmid DNA or even
100-10 ng for amplification of multicopy chromosomal
genes. For a cDNA synthesis reaction mixture, not exceed
10% of the final PCR reaction volume.
Quality control assays
Purity
NZYTaq DNA polymerase purity is >90% as judged by SDS
polyacrylamide gel electrophoresis followed by Coomassie
Blue staining.
Nuclease assays
0.2-0.3 µg of pNZY28 plasmid DNA are incubated with 1 µL
of 5× Gel Load Reaction Buffer in a 15 µL reaction for 14-16
hours at 37 °C. Following incubation, the DNA is visualised on
a GreenSafe-stained agarose gel. There must be no visible
nicking or cutting of the nucleic acid.
 Inadequate annealing temperature
The reaction mix composition may affect the melting
properties of primers and DNA. Adjust the annealing
temperature to accommodate the primer with the lowest
melting temperature (5 ° to 10 °C lower than Tm).
 Presence of PCR inhibitors
Some DNA isolation procedures, particularly genomic
DNA isolation, can result in the co-purification of PCR
inhibitors. Reduce the volume of template DNA in
reaction or dilute template DNA prior to adding to the
reaction. Diluting samples even 1:10,000 has been shown
to be effective in improving results, depending on initial
DNA concentration.
 Additives required
Adding PCR-enhancing agents (NZYTaq 5× Optimizer
Solution – MB060 or NZYTaq 2× GC-Enhancer Solution –
MB143) may improve yield while amplifying difficult
templates.
Revised 11/16
Certificate of Analysis
Test
Result
Enzyme purity*
Pass
DNase contamination
Pass
Functional assay
Pass
*These assays were exclusively performed with the enzyme
Approved by:
José Prates
Senior Manager, Quality Systems
Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E, R/C, 1649-038 Lisboa
Tel: +351.213643514 Fax: +351.217151168 www.nzytech.com